Bacteria, as is known, are the causative agents for a great many diseases in animals and humans. Before the advent of antibiotics, such as penicillin, bacterial infections were considered to be non-treatable. Since that time, however, the fight against bacterial diseases has often appeared to be won as antibiotic after antibiotic proved effective to combat the diseases and the bacteria that caused them.
Recently, however, bacterial diseases such as meningitis, pneumonia, tuberculosis, and enterotoxic diseases are on the increase due to the proliferation of antibiotic resistant strains of pathogenic bacteria. Bacterial resistance occurs because antibiotic therapy naturally kills most easily and swiftly the bacteria which are most sensitive to the antibiotic, leaving behind the bacteria which are less affected by the antibiotic therapy. Additionally, certain bacteria can pass antibiotic resistance genes to other otherwise sensitive bacteria. Over time, the populations of antibiotic sensitive bacteria disappear leaving only resistant populations.
This problem has been noted in scientific and popular articles, such as Begley, "The End of Antibiotics", Newsweek, Mar. 28, 1994, pages 47-52 ("Begley"), and Science, vol. 264, Apr. 15, 1994, the entire issue of which is dedicated to the problem of microbial resistance to antibiotics. Begley and Science vol. 264 are incorporated herein by reference.
Science has responded by discovering newer and better antibiotics with which to treat resistant bacteria. However, it appears that, as fast as new antibiotics can be produced, resistant strains of bacteria develop. Therefore, there is a clear and pressing need for new molecules and new means of treating bacterial infections.
Bacteria capable of producing disease in plants and animals, especially mammals, such as humans, and porcine, bovine, ovine, caprine, equine, feline, and canine species, such as pigs, cattle, sheep, goats, horses, cats, and dogs, require the production of certain proteins, known as virulence factors, or virulence determinants, to produce disease. That is, the bacteria are avirulent unless the virulence determinants are produced in an active form. Examples of virulence determinants include toxins, proteolytic enzymes, and structures such as pili which are required for adherence of the bacteria to tissues of the host organism. These virulence determinant proteins are generally secreted products, that is they are present on bacterial cell surfaces or are secreted totally outside of the bacterial cell. These exported virulence determinants pass through the bacterial periplasm on their way to their final location on or outside of the cell.
As taught in the parent application, a class of periplasmic bacterial oxidoreductase enzymes illustrated by the enzyme TcpG, have been discovered which function to catalyze the formation of disulfide bonds. The formation of these bonds allows the virulence determinant proteins to assume a functional, stable three dimensional conformation. Conversely, without these bonds, and the resulting active conformation, the virulence determinants are inactive. A major proportion of the teachings of the parent application has been published in a recent article by the inventors entitled "Characterization Of A Periplasmic Thiol:Disulfide Interchange Protein Required For The Functional Maturation Of Secreted Virulence Factors Of Vibrio Cholerae", PNAS; 89:6210-6214 (1992). This article is incorporated by reference and is cofiled with the present application as an integral part of this application.
The parent application teaches that preventing a microorganism from producing its oxidoreductase enzyme results in the production of inactive virulence determinants due to the lack of active 3-dimensional conformation. The parent application presents data showing that the lack of the periplasmic oxidoreductase enzyme TcpG in mutant Vibrio cholerae is responsible for failure of the mutants to produce active virulent cholera toxin.
In accordance with the invention, this application presents further data, set forth in an unpublished manuscript by the inventors entitled, "The Catalytic Site of Vibrio cholerae TcpG Disulfide Isomerase is Required for the Extracellular Localization or Function of a Variety of Secreted Virulence Factors." Further study has been made relating to the oxidoreductases' role in and relationship to te production of virulence determinant proteins and their symptoms. It has been found that the failure to produce active PcpG is responsible for the failure to produce active virulence determinant proteins, the effect of lack of TcpG is pleitropic, causing various effects on numerous virulence determinants which contain disulfide bonds, and that by providing active TcpG to bacteria incapable of producing TcpG, and therefore to produce inactive virulence determinant proteins.
There have also been developed several tests, in addition to the procedures taught in the parent application, to determine whether virulence determinants of a bacteria are active or inactive by measuring the symptoms thereof. By site-directed mutations that result in changing specific amino acids, the active site of the TcpG protein can be identified.
Periplasmic proteins with homologous structure and function to TcpG have been identified in other bacterial species, for example the DsbA protein in E. coli and the Por protein in Haemophilus influenzae, and similar homologous proteins identified in Salmonella typhimurium, Bacillus brevis, Legionella, and the plant pathogen Erwinia chrysanthemi. The identification of POREs in Bacillus suggests that POREs are likely present in other gram positive bacteria such as streptococcus and staphylococcus, important pathogens in humans and other animal species. These proteins, as well as other homologous proteins, already discovered or to be discovered in other bacterial species, are referred to collectively in this application as periplasmic oxidoreductase enzymes ("PORE"). However, neither the relationship of these PORE to the activity of virulence determinant proteins nor methods to inhibit the formation of active virulence determinant proteins by inhibition of the function of POREs have previously been disclosed.
Because of the extensive homology of structure and function of these proteins, it is believed, in accordance with the present invention, that observations related to structure and function.which are applicable to one member of the bacterial PORE family are applicable to all PORES. It has been discussed, for example, as described below, preventing the expression of the DsbA gene of E. coli results in the inability of the bacteria to produce an active pilus, rendering the bacteria avirulent. Likewise, preventing the expression of the TcpG gene in V. cholerae results in an avirulent bacteria with a morphologically normal but non-functional pilus.
Thus, it became apparent that inhibition of the function of POREs ultimately results in death of a pathogenic bacterium in vivo because of the failure of the organism to perform an essential function, such as the ability to colonize the intestinal wall.
Unlike antibiotics, however, inhibition of POREs does not result in lysis of the bacterial cell, which can contribute to the release of further endotoxins and exacerbate endotoxic shock as can occur in E. coli septicemia, for example.
The invention contributes to solving the serious problem of bacterial resistance to antibiotics. The invention provides a method for screening for and finding new antibacterial compounds which attack bacteria in previously unknown ways. The POREs taught herein represent a novel antibacterial drug target because, for one thing, until recently, POREs were not known to exist. Unlike most current antibiotics, which target the bacterial ribosomal protein synthesis apparatus or inhibit the formation of a functional bacterial cell wall, POREs are located in the periplasm and promote the production of active virulence determinant proteins. Thus, antibacterial compounds targeting POREs adds a new weapon in the fight against bacteria.
Antibacterial therapies aimed at POREs augments traditional antibiotic therapy because, as discussed below, many bacterial resistance proteins are secreted proteins which require POREs their activity in conferring resistance. It has been discovered that, the activity of bacterial proteins providing resistance to antibiotics, such as .beta.-lactamase which confers resistance to .beta.-lactam antibiotics such as penicillin and ampicillin, requires a functional PORE in order for the bacteria to be resistant to these antibiotics. Thus, the proteins which cause bacterial resistance to antibiotics can be rendered inactive by inhibiting POREs.
Additionally, therapies targeting POREs can be used in conjunction with conventional antibiotics to reduce the development of resistant organisms. The likelihood of developing resistance to multiple therapies aimed at different bacterial targets is much reduced compared with the development of resistance to only one therapeutic compound.
Moreover, it is believed that drugs which will inhibit PORE function are currently available, although their use in relation to PORE is not known. The reason is that, in screening molecules for antibacterial uses, pharmaceutical and biotechnology companies generally look for inhibition of cell growth or death of cells. However, inhibition of POREs in vitro does not inhibit cell growth nor cause cell death, but results in avirulent live cells. Thus, molecules which would be effective against virulent bacteria by inhibiting PORE function might well be missed by standard or conventional assays.
Additional background information is found in the articles listed in the bibliography of this application. The articles are incorporated herein by reference.